基孔肯雅病毒病毒样颗粒的制备及初步鉴定  

Preparation and identification of the Chikungunya virus virus-like particles

作　　者：陈何嵩[1] 程林峰[1] 马宏炜 叶伟[1] 韩佩君[1] 张亮[1] 雷迎峰[1] 张芳琳[1] 

机构地区：[1]第四军医大学微生物学教研室,陕西西安710032

出　　处：《热带医学杂志》2017年第3期285-288,F0004,共5页Journal of Tropical Medicine

摘　　要：目的通过杆状病毒表达系统表达制备基孔肯雅病毒(CHIKV)病毒样颗粒并进行初步鉴定。方法首先将基孔肯雅病毒的结构蛋白基因C-E3-E2-6K-E1克隆入p Fast-Bac^(TM) Dual转移载体中并鉴定;然后将构建正确的重组转移载体转化DH10Bac感受态细胞后提取重组杆粒并鉴定;再将重组杆粒转染Sf9昆虫细胞后获得重组杆状病毒,并利用免疫荧光法(IFA)鉴定其感染Sf9细胞后E2蛋白的表达;最后通过透射电镜(TEM)和Western-Blot鉴定CHIKV病毒样颗粒。结果成功获得了含有CHIKV结构蛋白基因的重组杆状病毒,该重组病毒经过IFA及WB验证在昆虫细胞中能够表达CHIKV的E蛋白,并在电镜下观察到了直径约60~80 nm的CHIKV病毒样颗粒。结论通过杆状病毒表达系统成功表达并获得CHIKV的病毒样颗粒,为CHIKV早期快速检测法的建立奠定基础。Objective To prepare and identify Chikungunya virus （CHIKV） viruslike particles with the baculovirus expression system. Methods The gene encoded the structural protein of Chikungunya virus C-E3-E2-6K-E1 was cloned into the pFastBac TM_ Dual vector. The recombinant DNA vector was confirmed by restriction enzyme digestion and DNA sequencing, and then transformed into E. coli DH10BacTM cells to isolate recombinant bacmid. The recombinant bacmid was confirmed by PCR amplification and then transfected into Sf9 insect cells to produce rBVs. IFA was used to identify the expression of E2 protein. Finally, TEM and Western-Blot were used to identify the expression of VLPs. Results The recombinant baculovirus containing CHIKV structural gene was successfully obtained. Further, the recombinant virus was confirmed by IFA and WB to be able to express E protein of CHIKV in insect cells. CHIKV virus-like particles with diameter of 60-80 nm were observed under electron microscope. Conclusion In this study, the expression of CHIKV VLPs by baculovirus expression system lays the foundation for the development of related Chikungunya virus rapid detection methods.

关 键 词：基孔肯雅病毒 杆状病毒表达系统 病毒样颗粒 

分 类 号：R373.3[医药卫生—病原生物学]